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目的:重组蛋白在表达和纯化过程中,甚至在存放过程中,都有可能产生降解、聚集,错误折叠、翻译后修饰(如:糖基化、氧化、脱酰胺),从而形成不同的表面电荷,影响产品的稳定性、活性和安全性。该研究旨在确定重组人纽兰格林原液中是否存在电荷变异体并对其定量。方法:采用极端的理化变性条件,制备重组人纽兰格林的电荷变异体;用HPLC(阳离子交换色谱柱,Tosoh Bioscience LLC,TOSOH TSK gelSP-STAT(4.6mm×10cm),7μm)分析重组人纽兰格林中的电荷变异体。结果:纯度99%以上的重组人纽兰格林原液,经酸破坏、高温条件下,产生了相对保留时间为1.1,质谱分子量为5786.28,N末端序列未发生变化的电荷变异体。重组人纽兰格林浓度在0.0156 mg/mL~1.25 mg/mL范围内,浓度与峰面积呈现良好的线性关系,y=17187x-309.09 R2=0.9998。专属性好:在流动相A、流动相B、水、重组人纽兰格林缓冲液中无重组人纽兰格林及电荷变异体峰。回收率:重组人纽兰格林在低(0.5mg/mL)、中(1.0mg/mL)、高(1.25mg/mL)3个浓度的回收率分别为99.0%、99.9%、100.7%。重复性:以重组人纽兰格林峰面积计算RSD为0.57%。最低检测限:电荷变异体的最低检测限为:0.16μg。最低定量限:电荷变异体的最低定量限为0.2μg。结论:该方法用于测定重组人纽兰格林电荷变异体,无杂质干扰,方法准确、可靠。
OBJECTIVE: Recombinant proteins may undergo degradation, aggregation, misfolding, post-translational modification (eg, glycosylation, oxidation, deamidation) during the process of expression and purification, even during storage, resulting in different surface charges , Affecting the stability, activity and safety of the product. The aim of this study was to determine if there is a charge variant in recombinant human neuregulin and quantify it. METHODS: Charged variants of recombinant neuregreen were prepared using extreme physicochemical denaturation conditions. Recombinant human neo were assayed by HPLC (cation exchange chromatography column, Tosoh Bioscience LLC, TOSOH TSK gel SP-STAT (4.6 mm × 10 cm), 7 μm) Charge Variant in Lange Green. Results: The recombinant human neuregulin with a purity of over 99% could produce a charge variant with a relative retention time of 1.1 and a mass spectrum molecular mass of 5786.28 without any change in the N-terminal sequence. The concentration of recombinant neuregulin in the range of 0.0156 mg / mL ~ 1.25 mg / mL showed a good linear relationship between concentration and peak area, y = 17187x-309.09 R2 = 0.9998. Good specificity: in the mobile phase A, mobile phase B, water, recombinant human neuregulin buffer without recombinant human neo-Neue Green and charge-variant peak. Recoveries: The recoveries of recombinant neuregulin were 99.0%, 99.9% and 100.7% at low (0.5mg / mL), medium (1.0mg / mL) and high (1.25mg / mL) concentrations respectively. Repeatability: The calculated RSD was 0.57% based on the area of recombinant neuregulin. Lowest detection limit: The lowest detection limit of charge variants is 0.16 μg. Lowest limit of quantification: The lowest limit of quantitation for charge variants is 0.2 μg. CONCLUSION: This method is suitable for the determination of recombinant human neuregulin charge with no impurity interference. The method is accurate and reliable.